We will continue to examine the nature of enzyme reaction mechanisms by use of reaction intermediates that are generated either in situ as for enolpyruvate, or in a single step by inactivation of a functioning enzyme as in the case of triose-P isomerase and metal dependent FDP aldolase. Other enzymes that we will examine are methylglyoxal synthase, which may produce the same intermediate as triose-P isomerase, and 6-P-gluconate dehydrogenase, which may produce the same enediol as D-pentose-P isomerase. By studying the pH dependence for reaction of enzyme with the intermediate it is possible to determine the contribution of diol and diolate forms to the reaction. The maximum rate of conversion of the proposed intermediate to normal products will be compared with normal Vmax of catalysis by the enzymes to establish the identity to normal intermediate. Other methods will be explored to determine the amount and chemical nature of intermediates that are too labile to stand by these methods.